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  • 1
    ISSN: 1546-1718
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Medicine
    Notes: [Auszug] Triple helix forming oligonucleotides (TFOs) recognize and bind sequences in duplex DNA and have received considerable attention because of their potential for targeting specific genomic sites. TFOs can deliver DNA reactive reagents to specific sequences in purified chromosomal DNA (ref. 4) and ...
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract A 21-bp deletion in the third exon of theAPRT gene in Chinese hamster ovary (CHO) cells was corrected by transfection with a plasmid containing hamsterAPRT sequences. Targeted correction frequencies in the range of 0.3–3.0×10−6 were obtained with a vector containing 3.2 kb ofAPRT sequence homology. To examine the influence of vector configuration on targeted gene correction, a double-strand break was introduced at one of two positions in the vector prior to transfection by calcium phosphate-DNA coprecipitation or electroporation. A double-strand break in the region ofAPRT homology contained in the vector produced an insertion-type vector, while placement of the break just outside the region of homology produced a replacement-type vector. Gene targeting with both linear vector configurations yielded equivalent ratios of targeted recombinants to nontargeted vector integrants; however, targeting with the two different vector configurations resulted in different distributions of targeted recombination products. Analysis of 66 independent APRT+ recombinant clones by Southern hybridization showed that targeting with the vector in a replacement-type configuration yielded fewer targeted integrants and more target gene convertants than did the integration vector configuration. Targeted recombination was about fivefold more efficient with electroporation than with calcium phosphate-DNA coprecipitation; however, both gene transfer methods produced similar distributions of targeted recombinants, which depended only on targeting vector configuration. Our results demonstrate that insertion-type and replacement-type gene targeting vectors produce similar overall targeting frequencies in gene correction experiments, but that vector configuration can significantly influence the yield of particular recombinant types.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Megakaryocyte differentiation is a lengthy process with cells moving through a continuum delineated by the sequential expression of specific gene products. The limited number of primary cells available from marrow for analysis has brought attention to some leukemic cell lines which show enhanced megakaryocyte marker expression following incubation with inducing agents, the most common of which is phorbol myristate acetate (PMA). We developed an alternative induction protocol for the megakaryocytic leukemic cell line CMK, which involved incubation of the cells with IL-3 and the nucleoside analog, ribavirin, for 1-2 weeks. This treatment was neither toxic nor cytostatic and yielded increased levels of the surface glycoproteins GPIIb/IIIA and GPIb-IX. Levels of some megakaryocytic messages (GPIIIa, GPIX) showed a marked rise by 12 days of incubation in the inducer combination. This was due to a synergistic interaction between IL-3 and ribavirin which influenced both transcriptional and posttranscriptional events. Light and electron microscopy demonstrated the presence of large polyploid cells, with morphological features similar to those of megakaryocytes, in the induced cultures. Analysis of the heterogeneity of response in the cell population to the induction regimen after several days of treatment suggested that cells which failed to display surface markers had been stimulated by the inducers but did not have sufficient time to complete expression of that marker. The results were consistent with the view that the cells in the starting population were distributed along a temporal expression pathway, and those which were first to express the earliest marker would also lead in the expression of a later marker. The order of expression was the same as that during normal megakaryocyte development. © 1994 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Differentiation of the megakaryocytic leukemia cells, CMK, was induced by long-term (12 day) treatment with the combination of IL-3 and the nucleoside analogue ribavirin (RV), which reduces cellular GTP levels. In a previous report we demonstrated the induction of early messages and antigens, as well as the formation of giant polyploid cells in the cultures (Majumdar et al., 1994, J. Cell. Physiol., 160:29-39). Here we show high level induction of messages for the late markers, Platelet Factor 4, GMP140 (P-Selectin), thrombospondin, and beta thromboglobulin. The induced cells are also positive for these antigens by immunocytochemical analysis. The high level message induction resulted from synergy between the inducers. Pretreatment of the cells with IL-3 could accelerate the rise in message seen with the inducer combination. The increase in differentiation markers was accompanied by a reduction of the proliferative capacity of the cells. Riboguanosine, which has anti differentiation activity, blocked the induction of early and late antigens by the inducer combination, and also by IL-3 acting alone, but did not block the reduction in proliferative competence. In this model of megakaryocytic differentiation IL-3 treatment yields and initial stimulation of growth followed by growth supperssion, and is the principal driver of the differentiation process. RV functions primarily as a stimulator of message and protein expression in synergy with IL-3. © 1995 Wiley-Liss Inc.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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